Editor’s Note: Scarcity of resources—money and time—keeps many researchers at home during scientific conferences. You can help them by sharing another critical resource—information. Meeting reports are an important way that you can provide a critical community service to many researchers toiling away in far-flung laboratories and offices, trying to piece together small patches of the schizophrenia puzzle. We encourage you to contact us if you would like to help spread the wealth that is information.

We are very fortunate to have C. Anthony (Tony) Altar as our guest correspondent for the Neuroscience 2008 meeting in Washington, DC. Tony is a unique researcher who keeps active collaborations going across academia, industry, and government.

3 December 2008. Presentations at the 2008 meeting of the Society for Neuroscience (SfN) provided support for the benefit of enhancing nicotinic and muscarinic cholinergic receptor signaling to improve schizophrenia, particularly the cognitive deficit and negative symptoms.

Nicotinic receptors and schizophrenia
We all know that nicotine elevates attention, memory, and cognitive performance, and that selective agonists for the α7 nicotinic receptor subunit are being tested for their ability to do this and more in schizophrenia and Alzheimer's disease. Unfortunately, the α7 nicotinic receptor partial agonist anabaseine (DMXB-A) failed to alter MATRICS measures or BPRS scores (Freedman et al., 2008; see SRF related news story). Before closing the doors on this approach, researchers continue working on several development programs in the nicotinic area. MEM 3454 is an α7 partial nicotinic receptor agonist and 5-HT3 antagonist. It improves cognitive functions in young adult and aged rats or mice. Mei Huang, Herb Meltzer, and colleagues at Vanderbilt University (abstract 874.23) worked with Memory Pharmaceuticals to evaluate MEM 3454 on cortical and hippocampal neurotransmitter release using microdialysis. Over a 0.1-10 mg/kg range of subcutaneous doses, MEM 3454 elevated dopamine (DA) and acetylcholine (ACh) release in the cortex and hippocampus, but not in the nucleus accumbens. MEM 3454 produced relatively small, ~40-60 percent overall increases in dopamine, and ~20-40 percent increases in acetylcholine, release, based on integrated area-under-the curve analysis, and an abrupt inverted U-shaped dose-response curve that peaked at the 0.5 mg/kg dose. Comparable increases in DA and ACh release produced by aripiprazole, risperidone, olanzapine, or quetiapine were augmented by a 0.45 mg/kg dose of MEM, but the effects of haloperidol were not. The MEM 3454 elevations of DA but not ACh release were blocked by the selective α7 receptor antagonist, MLA, whereas increases in ACh release but not DA release were blocked by the 5-HT3 antagonist, CPBG. Those straightforward studies show that MEM 3454 increases in DA and ACh release are mediated by α7 agonism and 5-HT3 antagonism, respectively, and are consistent with the improved cognitive function expected with this class of drug—potentially by itself, but as shown here, in combination with atypical antipsychotics. Drugs that possess nicotinic α7 agonism/5-HT3 antagonism may be useful adjuncts to existing antipsychotics, and represent receptor mechanisms that can be included in the design of drugs that block D2/5HT2A receptors. Anything like that out there?

It was also interesting to learn from Herb that his group has found comparable effects across many antipsychotic drugs on ACh and DA release in the hippocampus and frontal neocortex. Thus, you dialysists out there can study one or the other region, not both. They also mentioned that 0.3 to 0.5 mg/kg doses of Abilify produce the greatest increases in dopamine release in rat hippocampus or neocortex, with less effect at higher or lower doses. This translates well within the human dose of 2-20 mg per patient, which is pretty much the clinical dose range. Their findings support the use of hippocampal or cortical dopamine release, and not necessarily on both, for evaluating antipsychotic candidates and as one basis for selecting human doses.

Another α7 nicotinic agonist, WAY 317538, is a full agonist. As conducted in collaboration with researchers lucky enough to live in Siena, Italy (Siena Biotech), Wyeth researchers including Chad Beyer (abstract 657.16) showed that WAY 317538 more than doubled glutamate release in the rat medial frontal cortex and for at least three hours, without changing DA release. The lack of effect on DA may be due to its lack of H3 antagonism, which is a property of MEM 3454 (reviewed above). This glutamate-but-not-dopamine-release profile characterizes all α7 nicotinic agonists the Wyeth scientists discussed. Maybe it is for that reason that WAY 317538 was described as not going forward in development.

Amanda Williams and her colleagues at AstraZeneca showed a remarkable potency for the compound AZD0328. At 100 ng/kg, or only 20 ng per rat, AZD0328 improved performance in the cognitive memory task and novel object recognition test (abstracts 292.6, 906.23). Efficacy was lost at higher doses. When exposed to a long delay between the first and second test, most rats respond to a novel object with the same zeal and duration they would if they had never seen it. Even with the delay, AZD0328-treated rats spent only one-third of their time with the familiar object, and the rest of their time with the novel object. The report of a 67 percent decrease in α7 nicotinic receptors in frontal cortex and hippocampus after administration of AZD0328 is not surprising for nicotinic agonism, but warrants evaluations of its efficacy with chronic administration. This agonist is in clinical development at AstraZeneca in the areas of schizophrenia and Alzheimer's disease.

Another promising approach from AstraZeneca, in collaboration with Targacept, was reported by W.C. Moore and colleagues with AZD3480, an α2/β4/β2 nicotinic agonist (abstracts 329.4, 329.9). Moore described the use of this compound (formerly known as TC-1734) in Phase 2 trials for otherwise-unmedicated patients with schizophrenia. AZD3480 was studied 12 weeks after cholinergic deafferentation of the hippocampus by fimbria-fornix knife cuts. The degree of LTP, induced by stimulation of the entorhinal cortex in their in vitro hippocampal slice preparation, was measured by an enhanced EPSP response to subsequent stimulation. LTP was increased in tissues from intact rats by 60 nM AZD3480. This is very impressive, and the decrease in LTP produced by cholinergic lesion was restored to near-normal levels by AZD3480, and not 150 nM donepezil, which is not surprising since fornix lesion should leave little ACh for the donepezil to augment. These results may bode well for the use of the compound in illnesses like Alzheimer's disease, where cholinergic losses are well known, or for schizophrenia, where cholinergic inputs and signaling via muscarinic receptors may be diminished (as discussed in the even more exciting "Muscarinic" section of this report). Also promising was the efficacy of AZD3480 in the radial 8 arm maze and novel object recognition tests in intact animals. The behavioral efficacy of the compound in fimbria-fornix lesioned rats, or in those challenged with a low dose (like 0.3 mg/kg) of scopolamine, has not been evaluated, but it may be expected to work based on the LTP study.

Should investigators wish to confirm that their α7 nicotinic acid receptor-binding drugs actually get in the brain and bind to this target, [11C]CHIBA-1001 was reported at this meeting by Kenji Hashimoto of Chiba University (abstract 328.1) as the first and only PET ligand available for this purpose (Hashimoto et al., 2008). Some of the details of this method seemed less rosy. [11C]CHIBA-1001 shows only a 46 nM affinity for the α7 nicotinic acid receptor, defined by [125I]α-bungarotoxin, and was only tested at 28 other receptors for inactivity. Seemingly too much of the label (75 percent) persisted in rhesus macaque brain even when co-treated with 5 mg/kg of unlabeled SSR 180711, an α7 nicotinic receptor agonist. No [11C]CHIBA-1001 label was displaced with an α4β2 nicotinic agonist. The PET signal persisted for two hours post-intravenous infusion, but the high degree of non-displaceable labeling is a concern. Fortunately, in human brain, Masatomo Ishikawa, also at Chiba University, showed binding to be more (~50 percent) specific in healthy subjects, and visibly displaceable occupancy in the neocortex and cerebellum was obtained with a 10 mg dose of a "proprietary" α7 nicotinic agonist (abstract 328.2). Hopefully that agonist was something different from CHIBA-1001 itself.

Muscarinic receptors and schizophrenia
While clinical proof of concept for muscarinic agonism has been provided for treating cognitive deficits of schizophrenia (Shekhar et al., 2008) and the delusions and hallucinations of Alzheimer's disease patients (Bodick et al., 1997), a deficiency in muscarinic transmission that may subserve this approach has been more clearly provided at this year’s meeting. Studies by Holt and colleagues showed decreases in cholinergic innervation of the nucleus accumbens in schizophrenia (Holt et al., 2005; Holt et al., 1999). Modeling this loss in rats with bilateral accumbens infusions of the selective cholinergic toxin, Francois Laplante and colleagues from the University of Montreal used saporin-anti-ChAT IgG immunoglobulin to deplete 50 percent of ACh neurons in this region (abstract 761.18). Though selective, this procedure did not change basal startle but decreased prepulse inhibition of startle by up to 40 percent, and slightly increased basal locomotion, grooming, and "other" movements. Some of these changes were increased by the D2 agonist quinpirole. These findings support the old-as-haldol DA/ACh balance hypothesis by showing that increases in this ratio predispose to psychosis (or psychosis-related behaviors in animals), and, equally important, that decreases in the ratio—by blocking D2 receptors or increasing cholinergic transmission—can treat the disease.

More support for the benefit of augmenting muscarinic cholinergic tone in schizophrenia was obtained by Hasib Salah-Uddin, Jeannette Watson, Brian Dean, and colleagues at the University of Leicester, GlaxoSmithKline, and the Mental Health Research Institute, Australia (abstract 54.19). They provided compelling new support for altered muscarinic receptor signaling in schizophrenia. Prior reports of decreased muscarinic receptors in the cortex, caudate putamen, and entorhinal cortex in schizophrenia by Dean and colleagues (Dean et al., 2000; Crook et al., 2001) are impressive enough, but do those decreases translate to a decrease in muscarinic signaling? Using postmortem dorsolateral prefrontal cortex (DLPFC), from schizophrenia and control subjects, they have shown that about 25 percent of people with schizophrenia have a ~70 percent reduction in [3H]-pirenzepine binding. This group has been termed "muscarinic receptor-deficit schizophrenia" (MRDS) (Scarr et al., 2008). The MRDS subgroup in the new study showed not only the ~70 percent decrease in [3H]-pirenzepine binding, but a 0.5 log unit right-shift in the potency of the orthosteric muscarinic receptor agonist oxotremorine-M in stimulating [35S]GTPγS binding in membranes prepared from these brains. About a 20 percent greater degree of [35S]GTPγS binding, stimulated by the orthosteric agonist, also characterized the MRDS patients. The functional activity of the allosteric site agonist, AC-42, was not affected in the MRDS population. The potency and relative efficacy of M1 receptor-agonist coupling for oxotremorine-M and AC-42 was similar for controls and non-MRDS subgroups. These findings combine nicely with deficient expression of genes in hippocampal neurons of schizophrenia patients (Altar et al., 2005), which could be reversed by oxotremorine-M in human neuronal cell line (Altar et al., 2008). Many of those gene decreases in schizophrenia were replicated in a second cohort of patients, and were consistent with a metabolic deficit hypothesis of schizophrenia.

As long as we are on the topic of a metabolic deficit in schizophrenia, Rosalinda Roberts, newly ensconced at the University of Alabama, and her colleagues (abstract 656.6) found 30 percent less cytochrome oxidase (COX; p <0.001) in the putamen of schizophrenia cases versus normal controls, and these were lower regardless of whether the schizophrenia patients were on or off medications at the time of death. Also arguing against medication status in these effects was their finding in rats that COX activity was not decreased by haloperidol or clozapine compared to vehicle-treated controls. The decrease in COX activity in the putamen of schizophrenia cases agrees with prior reports by others including those of Prince and colleagues (see, e.g., Prince and Oreland, 1998), and may be due to mitochondrial abnormalities in the putamen, a possibility that is supported by the diminished number or size of mitochondria in schizophrenia cases also reported by this group. It might be of interest for Rosie to see if there is an MRDS-like segregation in these cases and if so, whether they co-segregate with deficient muscarinic receptor coupling to [35S]GTPγS binding.

While muscarinic receptors have been known to facilitate long-term potentiation (LTP) in the hippocampus, muscarinic receptor subtypes responsible for this were unknown. Katherine Buchanan of University College, London, used the drug 77-LH-28-1, a selective allosteric M1 muscarinic acetylcholine receptor agonist, to show that M1 activation facilitates LTP in the young rat hippocampus (abstract 335.11). When applied to hippocampal slice preparations maintained in vitro, 77-LH-28-1 depolarized and increased membrane resistance in CA1 neurons, but did so without changing glutamate release. In response to "theta burst stimulation,” 77-LH-28-1 increased LTP due to glutamate release, and a similar LTP increase by 77-LH-28-1 was produced in response to increased endogenous ACh release from stimulated cholinergic afferents, or by the non-selective, non-hydrolyzable cholinergic agonist, carbachol. Their work demonstrates a mechanism by which M1 acetylcholine receptors can amplify LTP induction in the hippocampus and thereby enhance cognitive function. This would represent another example of tonic amplification of cholinergic stimulation, whose attractiveness lies in the selectivity it imparts during heightened cholinergic transmission.

That the entorhinal cortex itself may contribute to a habituation impairment in schizophrenia was suggested by the findings of Segev Barak and Ina Weiner of Tel Aviv University (abstract 418.3). They noted that subjects with schizophrenia have a deficiency in latent inhibition (LI), as they are more likely to attend to irrelevant stimuli presented during preconditioning trials. As shown previously with systemic injection of the muscarinic antagonist scopolamine, bilateral infusion of 1-10 μg scopolamine into the rat entorhinal cortex disrupted LI. This showed that at least in this region, muscarinic receptor agonism supports the ability to inattend to irrelevant stimuli. That feature of inattention is one in which subjects with schizophrenia do poorly on. Barak and Weiner also showed that muscarinic receptor agonism in the amygdala promoted a re-attending to previously irrelevant stimuli that are now relevant. Thus, like the Buchanan study with LTP described in the previous paragraph, known roles for cholinergic function in LTP or attention mechanisms have now been shown to be mediated by muscarinic receptors. Both the LTP and LI models can be considered as tests for drugs designed to work through muscarinic receptor agonism, although the potentially counterproductive role of amygdala muscarinic agonism may be problematic for muscarinic agonist therapeutics.

While the desired preference of muscarinic agonists for M1/3/5 over M2/4, and for M1 over other receptors, is likely for CNS targeting and for avoiding peripheral cholinergic side effects, the design of selective compounds for these targets is challenging. Muscarinic receptors are highly conserved in the vicinity of the acetylcholine orthosteric binding site. John Ellis and Edward Stahl of Penn State University showed that muscarinic receptors possess allosteric sites, at which receptor activity may be positively or negatively modulated (abstract 532.5). They reported that amiodarone enhances the activity of ACh at the M5 receptor relative to M1, probably by binding within extracellular loop(s) of the receptor.

Ditte Dencker and Anders Fink-Jensen of the University of Copenhagen used mice produced by Jongrey Jeon (NIH), in which an elegant conditional inactivation of mouse M4 muscarinic cholinergic receptors is produced when those receptors reside on DAD1-receptor-containing striatonigral GABA neurons, and medium spiny striatal cholinergic interneurons (abstract 743.2). These mice showed a 75 percent attenuation of 0.3 mg/kg haloperidol catalepsy, and nearly as much loss at a whopping 1 mg/kg haloperidol dose. Even a 5 mg/kg dose of the non-selective muscarinic receptor blocker, scopolamine, was unable to block what little haldol catalepsy remained in the M4-deletion mice. Thus, the EPS side effects of typical antipsychotics like haloperidol appear to be promoted by endogenous cholinergic activation of M4 receptors on D1 receptor-containing neurons. Their findings are consistent with the use of anticholinergic drugs to block EPS, and refine this to suggest that a centrally selective M4 antagonist/M1 agonist might be a useful adjunct for treating Parkinson's disease or the EPS side effects associated with typical antipsychotics, while simultaneously improving cognition. Now that's a drug discovery challenge!—C. Anthony Altar.

Firstly, I'd like to say how much I appreciated being able to get a precis on my area of interest from a conference I was unable to attend—it's a great concept. Thanks to Tony for producing it.

In answer to the question as to whether there is a CHRNA7 agonist/5-HT3 antagonist available: Tropisetron is such a compound. It was shown to reduce P50 deficits in 17 of 19 patients with schizophrenia (Koike et al., 2005). The authors made no reference to any other parameters and I haven't heard of any other studies using it.

Before Christmas, an insightful discussion between SRF's Pete Farley and researchers Heather Mefford and Evan Eichler delved into the complex interplay between genotype (copy number variant status at 1q21.1) and phenotype (psychiatric illness, autism, mental retardation, and congenital abnormalities) (see SRF related news story). The upshot was that although deletions at this locus were statistically associated with pathologies, the severity and nature of those pathologies was extremely variable. This raised questions about whether researchers and clinicians should focus on the disease or the deletion, and what the mechanisms that determine the clinical endpoint might be. This is becoming a clear trend. Another CNV region at 16p11.2 has also been variously associated with both autism and schizophrenia. Deletions of just a single gene, CNTNAP2, as opposed to a gene cluster, have also shown this phenomenon of variable phenotype expression—deletion carriers have been diagnosed with autism, Gilles de la Tourette/obsessive compulsive disorder, schizophrenia/epilepsy, or remain entirely healthy (Bakkaloglu et al., 2008; Friedman et al., 2008; Verkerk et al., 2003; Belloso et al., 2007).

In the same vein, this new paper by Helbig and colleagues describes yet another example of a discrete copy number variant (microdeletion) that was originally linked with psychiatric phenotypes but is now also shown to give rise to idiopathic generalized epilepsy (IGE). The deletion is at 15q13.3, which encompasses the candidate neurotransmitter receptor gene, CHRNA7, among others. In fact, with a frequency of 1 percent in the IGE population and absence in controls, the deletion is the strongest genetic risk factor for this condition and is more prevalent in IGE than in either mental retardation or schizophrenia.

Although the study of CNVs has highlighted this genotype-phenotype issue, it has been observed previously in the context of the overlap of linkage hotspots between schizophrenia and bipolar disorder (Berrettini, 2003), in case-control association studies linking the same gene to multiple disorders (Chubb et al., 2008), and in the case of the Scottish family with the t(1;11) translocation disrupting DISC1, in which carrier phenotypes ranged from healthy to major depression, bipolar disorder, and schizophrenia (Blackwood et al., 2001).

So we are now faced with complex genetic disorders that really live up to their name. As such, two particular issues warrant further discussion.

The first issue is that clinicians seem to observe discrete rather than continuous disorder phenotypes. Despite the current diagnostic manuals leaving little room for diagnostic leeway, it seems that the majority of case phenotypes tend toward a limited number of outcomes such as schizophrenia, bipolar disorder, mental retardation, autism, and epilepsy. Moreover, no psychiatrist can distinguish DISC1 schizophrenia from 1q21.1 schizophrenia or NRG1 schizophrenia without recourse to genetic methodologies, suggesting that there is a positive biological drive towards the endpoint. To borrow what may be a useful analogy from physics, the system is “chaotic” (in terms of its genetic input and its effect on cellular biology) but tends toward “strange attractors” (a limited set of diagnoses) [http://en.wikipedia.org/wiki/Attractor]. Why might this be so? It may be that there are several higher order functional bottlenecks within the brain such as synaptic transmission efficiency, cortical development, astrocyte/oligodendrocyte function, hippocampal neurogenesis, higher order communication between brain regions, etc. These act to “sum” the expected environmental, genetic, and cellular complexity present within an individual and transform it into a limited set of potential outcomes—in essence, these are the strange attractors.

The next issue is how the same mutation can give rise to two (or more) different conditions. It may be useful to think of the Knudson “two-hit” hypothesis of cancer in which environment and other genetic factors act subsequent to a “deep” genetic fault (Knudson, 1971).

The CNV examples above may represent such fundamental disruptions and most probably impinge on neurodevelopmental pathways, priming the brain to be tipped over the threshold into a disease state. In fact, the t(1:11) translocation carriers present evidence for such a phenomenon as both healthy and affected carriers show abnormal P300 brain response activities suggesting this endophenotype highlights an underlying brain dysfunction (Blackwood et al., 2001).

We have to postulate that the additional genetic or environmental influences (modifiers) not only determine entry into the disease state but also dictate the final outcome. Possible candidates for modifiers of the deletions above are the remaining single copy alleles at the CNV locus—exposed recessive mutations, imprinting, or epigenetic modification could all alter expressivity and penetrance of the deletion phenotype. However, limited studies by Eichler’s group seem to discount this possibility (Mefford et al., 2008).

In any case, genomewide association and CNV studies suggest that there is plenty of scope for a sufficient burden of genetic modifiers outside the CNV region. This may also fit in with the seemingly disparate concepts of rare/familial variants exposed by linkage and common/low odds ratio variants revealed by association. Both act causally with the former potentially acting as the “first hit.”

As time progresses, we will move towards the definition of the range of phenotypes potentially resulting from each genotype and the spectrum of genotypes causing each phenotype. CNVs represent a pretty blunt tool to dissect finer relationships between genotype and phenotype, so it is to be expected that rare but penetrant point mutations that emerge from resequencing projects will be of greater use in dissecting function-phenotype links—as has been seen with the connexin gene family, for example (Rabionet et al., 2002).

In summary, it is to be hoped that the clinical and research communities are able to embrace these complexities for what they offer—a deeper understanding of these disorders, one that is intimately linked to the development and function of the brain.